Modulation of Kex2p Cleavage Site for In Vitro Processing of Recombinant Proteins Produced by Saccharomyces cerevisiae

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P1', P2', P4, and P3 sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.


Introduction
Yeast is a popular eukaryotic host for recombinant protein production because it can be easily cultured to high cell densities and efficiently secrete proteins after post-translational modification similar to those in higher eukaryotic cells [1,2].Secretory production of recombinant proteins fused with fusion partners and in vitro processing with sequence specific endo-proteases is favored because it greatly simplifies the purification procedure compared with intracellular production.For this purpose, Factor Xa protease and enterokinase (EK) are generally used because they can produce intact target proteins when fusion partners are fused to the Nterminus of target proteins.However, they frequently cleave at other nonspecific basic residues [3][4][5] and cost of these proteases is too high to use in large-scale production of recombinant proteins.
The product of Saccharomyces cerevisiae KEX2 (Kex2p, E.C. 3.4.21.61) is a Ca 2+ -dependent membrane-bound serine protease [6,7].Kex2p matures K1 killer toxin [8] and mating factor a (MF) [9] prohormones by cleaving after dibasic residues (Lys-Arg and Arg-Arg) in the late Golgi network.Because dibasic residues are frequently found in proteins, exceptionally high substrate specificity is required for Kex2p to discriminate non-substrate proteins [10].These characteristics imply that substrate specificity of Kex2p can be modulated by changing the amino acids adjacent to the cleavage site [11][12][13].In addition, Kex2p is highly efficient because this membranebound enzyme is shortly exposed to correct substrate proteins mixed with a lot of non-substrate proteins in the trans-Golgi network.This process is very similar to the processing of mammalian pro-proteins by mammalian homologues of Kex2p [for example, furin and pro-protein convertase family (PCs)], implying conservation of this process throughout eukaryotic evolution [14].Kex2p cleaves several mammalian pro-proteins, such as human pro-albumin [15], protein C precursor [16], and insulin-like growth factor-1 [17].
Many studies have been conducted to exploit these advantages for the production of recombinant proteins.When the amount of recombinant proteins exceeds processing capacity of Kex2p in yeast, overexpression of KEX2 enhances the secretion of recombinant proteins [18].In addition, modification of the P 1  site to induce exposed loop structure of Kex2p recognition site led to the enhanced secretion of target proteins in Pichia pastoris [19].Accordingly, soluble Kex2p variants have been produced by truncating the transmembrane (TM) domain in S. cerevisiae [20][21][22] and applied for in vitro processing of fusion proteins [20].To produce fusion proteins joined by the Kex2p cleavage sequence without in vivo processing in yeast, a kex2 mutant strain should be used.Although KEX2 is not an essential gene, the null mutant is very sensitive to various stresses, and growth in complex media is severely retarded.Therefore, the kex2 mutant strain was not an appropriate host for recombinant protein expression.When other expression systems are used to express recombinant proteins, such as bacteria and cell lines, Kex2p can be used without considering the kex2 mutant.
In this study, instead of using kex2 mutant strain, Kex2p cleavage sites of fusion proteins were elaborately manipulated using the ProP 1.0 server, which predicts cleavage sites for furin and PCs to enhance or repress in vivo processing in yeast and to guarantee in vitro processing with Kex2p.

Strains, Chemicals, Media, and Analysis of Secreted Proteins
Escherichia coli DH5 [F− lacZΔ M15 hsdR17(r-m-) gyrA36] was used for the plasmid constructions.S. cerevisiae allgal (Mat α pep4::HIS3 prb1, can1, his3-200, gal80, gal1, gal2, gal7, gal10) [23] was used as a general host for recombinant protein expression.Q5 High-Fidelity DNA Polymerase and restriction endonucleases were purchased from New England Biolabs (USA) and used for genetic manipulation.DNA purification was performed using Wizard SV Gel and the PCR Clean-Up System (Promega, USA).Yeast transformation was performed using the lithium acetate method [24].Yeast transformants were selected by cultivation at 30°C in synthetic defined medium lacking uracil (SD-URA; 0.67% yeast nitrogen base without amino acids, 2% glucose, 0.77 g/l -uracil dropout supplement, and 2% agar).YPD medium (1% yeast extract, 2% peptone, and 2% glucose) was used to express recombinant proteins.Transformants were cultivated in a 10-ml test tube containing 2 ml of YPD broth medium for 40 h with vigorous shaking.Subsequently, 0.6 ml of culture supernatant was precipitated by centrifugation at 10,000 g for 20 min with 0.4 ml of cold acetone and analyzed using SDS-PAGE on 12% polyacrylamide gels (Bio-Rad, USA) under denaturing conditions and then stained with Coomassie blue.To determine the molecular weight of glycoproteins after deglycosylation, precipitated culture supernatants were treated with endoglycosidase H (Promega).

Fermentation and Protein Purification
For the fermentation of recombinant strains, seed culture was prepared using 200 ml of SD-URA broth contained in a 1,000-ml Erlenmeyer flask, and incubated overnight at 30°C.The cultured seed (200 ml) was inoculated into a 5-L jar fermentor (Kobiotech, Korea) containing 1.8 L of medium consisting of 2% glucose, 40 g yeast extract, and 10 g peptone (per liter).After glucose consumption, a feeding medium containing 300 g glucose, 300 g galactose, and 150 g yeast extract (per liter) was added.The hourly feeding rate was manually increased from 2 to 10 g/l according to cell growth.The pH (5.5) was controlled using NH 3 (25%).For the analysis of secreted proteins from fed-batch fermentation, 10 l of culture supernatant was directly used for SDS-PAGE after mixing with 2×SDS-PAGE sample buffer.
HL-hEGF proteins were purified by immobilized metal-affinity chromatography (IMAC) on a nickel-NTA agarose column (Promega) using a low-pressure liquid chromatography (Bio-Rad HR system).Fermentation broth was filtered using 0.1 m Sartoclear (Sartorius AG, Germany), concentrated five times using 30,000 NMWC Quick-stand (Amersham-Pharmacia Biotech, USA), and adjusted to buffer A [50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl].After loading onto the column at a flow rate of 1 ml/min, the proteins were eluted in a gradient of buffer B [50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 0.5 M imidazole].The fractions containing the protein of interest were concentrated and exchanged into Kex2p working buffer [50 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 2 mM CaCl 2 ] using a 10,000 MWCO Amicon Ultra centrifugal filter device (Millipore, USA).The HL-tag was separated by digestion with CaKex2p [22], which was prepared in-house.One microgram of CaKex2p was used per 1 mg of fusion protein and was incubated at 30°C for 1 h.Intact hEGF was obtained by repeated metal-affinity chromatography on a nickel-NTA agarose column using low-pressure liquid chromatography.The molecular weights of the purified proteins were determined using ProteinWorks (Korea).

Analysis of hEGF Activity
The bioactivity of hEGF was determined by proliferation assays using HaCaT cells.Cells were cultured with growth media [DMEM (Thermo Fisher, USA) + 10% FBS (Thermo Fisher) + 100 u/ml Penicillin-Streptomycin (Thermo Fisher)] in a humidified incubator containing 5% CO 2 at 37°C.After cultivation to 80-90% confluence in T75 culture flasks, the medium was replenished with DMEM + 100u/ml Penicillin-Streptomycin supplemented with different concentrations of hEGF and cultured for 48 h before harvesting.After removing the growth media, the cells were incubated for 1 h at 37°C with Ez-Cytox solution (Daeil Lab Service Co Ltd., Korea).Absorbance was measured at 490 nm using a microplate reader (Molecular devices, USA).Statistical comparison of growth was performed using the Student's t-test with a two-tailed distribution (Microsoft Excel).Values were considered statistically significant at p value of < 0.05.

Secretory Expression of hEGF with HL Fusion Partner in S. cerevisiae
When hEGF was expressed with the hydrophilic 28-amino acid peptide (HL28 fusion partner) from Voa1p using the YEG-HL28-hEGF vector under the control of GAL10 promoter [25] in S. cerevisiae, although the expression of hEGF was greatly improved, a large amount of unprocessed MF pro-H28L-hEGF fusion protein was detected in the culture broth as smear protein bands due to the hyper-mannosylation with varying extents (Fig. 1B, lane 1).HL28-hEGF does not contain a glycosylation site and the MF pro-peptide contains three glycosylation sites; therefore, secretion of hyper-glycosylated fusion proteins means processing of the MF pro-peptide by Kex2p in the Golgi network was incomplete.Since the substrate specificity of Kex2p is related to amino acids adjacent to the dibasic cleavage site [11,12], to increase processing of MF pro-peptide by Kex2p, the P 1  and P 2  positions, i.e. the N-terminus of the HL domain was modified using the ProP 1.0 server (https:// services.healthtech.dtu.dk/service.php?ProP-1.0)[27], which predicts pro-peptide cleavage sites in eukaryotic protein sequences using the furin and PCs neural networks.If the ProP score is >0.5, the residue is predicted to be a pro-peptide cleavage site, and a higher ProP score is more confident in the prediction.Although the substrate specificity of furin is slightly different from that of Kex2p, using this program, serine and valine were selected as the P 1  and P 2  residues that showed the highest ProP score, regardless of P 3  and P 4  residues.After the addition of Ser-Val to the N-terminus of the HL domain, the ProP score increased from 0.220 to 0.567.The expression of the modified vector (YEG-HL18-hEGF) was compared with that of the original vector (YEG-HL28-hEGF) by SDS-PAGE of culture supernatants of transformed yeasts.The hyper-glycosylated MF pro-HL-hEGF fusion proteins expressed from the modified vector were significantly decreased compared to the original vector (compare smear bands of lanes 1 and 5 of Fig. 1B) and this decrease was clearly demonstrated after deglycosylation (compare pro-HL-hEGF bands of lanes 2 and 6 of Fig. 1B) and after treatment with Kex2p (Fig. 1B, lanes 3 and 7), implying increased processing of the MF pro-peptide by Kex2p in the Golgi complex.The HL fusion partners were easily separated from the fusion proteins by digestion with EK (Fig. 1B, lanes 4 and 8).

Engineering of Kex2p Cleavage Sites for In Vitro Processing of Fusion Proteins
Although EK is a generally used endo-protease for the processing of fusion proteins, its application is restricted in laboratories because of nonspecific cleavage at other basic residues and high cost.First, to use Kex2p instead of EK for the in vitro processing of hEGF fusion proteins, a Kex2p cleavage site was generated by adding Arg just behind the EK cleavage site (DDDDKR, ProP 0.162).However, the hEGF fusion protein was not processed despite prolonged incubation with excess Kex2p (data not shown).Therefore, the P 3 and P 4 sites, instead of the P 1  and P 2  positions of the newly introduced Kex2p cleavage sequence, were manipulated by adding three residues at the P 3, P 4 and P 5 sites based on ProP 1.0 analysis because the modification of the P 1  and P 2  sites changed the N-terminus of hEGF.Four kinds of expression vector variables at P 3 and P 4 sites were constructed, and the expression of hEGF fusion proteins was compared with that of YEG-HL18-hEGF constructed previously.ProP scores were adjusted to 0.167-0.695by changing the P3 and P4 sites (Fig. 2B).The amount of secreted hEGF fusion proteins was slightly affected by the modification of linker sequences, but Kex2p processing was significantly influenced by the P3 and P4 residues.All variants were secreted as HL-hEGF fusion proteins without in vivo processing by Kex2p (Fig. 2C, lanes 2-5).To confirm in vitro processing of HL-hEGF fusion proteins by Kex2p, Kex2p digested protein bands (Fig. 2C, lanes 7-10) were compared with HL18-hEGF digested with EK (Fig. 2C, lane 6).Unlike other variants, the HK1-hEGF variant (ProP 0.167) was not cleaved by Kex2p, and with the increase in ProP score, HK2-hEGF, HK3-hEGF, and HK4-hEGF were partially processed with Kex2p (compare Fig. 2C, lanes 7 and 8-10).Although the HL tags liberated from HK2-hEGF, HK3-hEGF, and HK4-hEGF by digestion with Kex2p were larger by 5 amino acids than that from the HL18-hEGF digested with EK, they were not identified on SDS-PAGE, contrary to HL fusion partners digested with EK (Fig. 2C, compare lanes 6 and 8-10).Therefore, when the HL-hEGF fusion proteins were digested with Kex2p, the HL fusion partner appeared to be further processed into small peptides.Ultimately, to block the in vivo processing and to allow the in vitro processing of the HL-hEGF fusion protein by Kex2p, the ProP score should be higher than 0.169.

Production of Intact hEGF Using In Vitro Processing by Kex2p
To confirm in vivo processing during prolonged cultivation and large-scale production of intact hEGF by in vitro processing with Kex2p, fed-batch fermentations of recombinant S. cerevisiae 2805 allgal strains expressing three HL-hEGF variants were conducted.The growth of the three strains reached 120-130 OD 600 similarly after 48 h of fermentation, but the amount and pattern of secreted HL-hEGF fusion proteins were significantly different.HK2-hEGF was produced as a fusion protein without in vivo processing, similar to the test tube culture (Fig. 3A), but HK3-hEGF was partially processed after 24 h of fermentation and HK4-hEGF was more significantly processed than HK3-hEGF (Figs. 3B and 3C).HK4-hEGF was produced as a mixture of fusion proteins and hEGF.The smaller protein band than that of HK4-hEGF seemed to be a misprocessed fusion protein at the P 3 position of the linker because the P 4 and P 3 sites of HK4-hEGF are dibasic residues (Arg-Lys).The HK2-hEGF fusion protein was purified by IMAC using A poly-histidine tag and then digested with Kex2p to remove the HL fusion partner.HK2-hEGF was completely digested to hEGF and intact hEGF was easily obtained by the 2 nd IMAC (Fig. 3D).Purified hEGF demonstrated similar growth-stimulating activities to commercial hEGF (SIGMA) in the growth of HaCaT cells (Fig. 3E).Because the affinity tag is contained in the HL fusion partner, hEGF processed during secretion is not captured by IMAC; therefore, the recovery yield of HK2-hEGF was higher than those of HK3-hEGF and HK4-hEGF at 1 st IMAC.Finally, the recovery of intact hEGF from HK2-hEGF was more than two-fold higher than those from HK3-hEGF and HK4-hEGF (Table 1).Therefore, to completely block the in vivo processing of the HL-hEGF fusion protein by Kex2p during fermentation, the ProP score should be lower than that of HK3-hEGF (0.189).

General Application and Optimization of In Vitro Processing by Kex2p
To confirm the general application of Kex2p for the in vitro processing of fusion proteins in yeast, phytase, exendin-4, and defensin-2 were expressed with HL fusion partners.Phytase is a phosphatase that catalyzes the hydrolysis of phytic acids found in many plant tissues.Exendin-4 is a potent glucagon-like peptide-1 receptor agonist that promotes insulin secretion, and defensins are host defense peptides (typically 18-45 amino acids long) with antimicrobial activity against bacteria and fungi.Phytase from Citrobacter braakii (CbPHY) [28], exendin-4 from Heloderma suspectum (EXD4) [29] and defensin-2 from Zea mays (ZmBD2) [30] were expressed with HL fusion partners, and the ProP score of Kex2p cleavage sites between HL fusion partner and target proteins was adjusted to 0.157-0.223(CbPhy: 0.159-0.191,EXD4: 0.183-0.216,and ZmBD2: 0.195-0.223)by modifying the P 3 and P 4 sites.The expression of CbPhy and ZmBD2 was slightly enhanced by tagging with HL (Fig. 4A and  4C), whereas EXD4 was expressed only when HL was fused (Fig. 4B).In the case of CbPhy, even though the ProP score of HK2-CbPhy (0.157) was lower than that of HK1-EGF (0.169, Fig. 2B), a significant amount of fusion proteins was in vivo processed by Kex2p.In contrast, the ProP score of HK3-EXD4 (0.216) was higher than that of HK3-EGF (0.189, Fig. 2B), and in vivo processing did not occur.In case of HL-ZmBD2, in vivo Kex2p processing was controlled as expected.HK1-ZmBD2 and HK7-ZmBD2 were expressed without in vivo processing, and most of fusion proteins were processed by incubation with Kex2p.These results indicate that Kex2p processing does not correctly correspond to the ProP score according to the target proteins but can be optimized by adjusting the ProP score to approximately 0.15-0.20,by modulation of the P 3 and P 4 sites.

Fig. 4 .
Fig. 4. In vitro processing of fusion proteins containing engineered linkers.SDS-PAGE analysis of culture supernatant of (A) HL-CbPhy, (B) HL-EXD4, and (C) HL-ZmBD2, with and without Kex2p digestion.Structure of fusion proteins are indicated.C: proteins expressed without HL fusion partner.The corresponding protein bands are indicated by arrows.M: protein molecular weight markers.(D) Amino acid sequence and ProP score of the modified linkers.